High Efficiency Yeast Transformation

Materials

Transformation Mix (tMIX)

Reagent X1 X5 X10 X15 X20 50% PEG 4000 240 ?l 1200 ?l 2400 ?l 3600 ?l 4800 ?l 1M LiAc 36 ?l 180 ?l 360 ?l 540 ?l 720 ?l 10 mg/ml ssDNA 10 ?l 50 ?l 100 ?l 150 ?l 200 ?l H2O 74 ?l 370 ?l 740 ?l 1110 ?l 1480 ?l --- --- --- --- --- --- Final Volume 360 ?l 1800 ?l 3600 ?l 5400 ?l 7200 ?l

ssDNA must be freshly denatured every time by boiling at 95°C for 10 minutes

Method

Start from exponentially growing cells at a concentration of 5?106: 10 ml for each transformation reaction;
Prepare the required amount of tMIX
Pellet 50 ml (=for 5 transformations) of cells 4' in a 50 ml tube at 4000 rpm at room temperature;
Wash the cells with 25 ml of sterile water;
Resuspend the pellet in 1 ml of sterile water and transfer to 2 ml tubes;
Spin and resuspend in 500 ?l (100 ?l for each transformation);
For each transformation, transfer 100 ?l of cell suspension in a new 1.5 ml tube, ;
Spin and discard the supernatant;
Add 360 ?l of Tmix (freshly prepared);
Add DNA:
- 200-500 ng for plasmids (1-4 ?l of a mini prep);
- 2-3 ?g for deletion/tag cassette (5-10 ?l of precipitated PCRs);
Resuspend the cells using a vortex;
Incubate at 42°C:
- 5'-10' for plasmids;
- 20-25' for multiple plasmid transformations;
- 40' for transformations that requires a recombination event (e.g. cassettes, gap repair);
Pellet the cells at 4000 rpm for 1' and discard the supernatant;
If the transformation involves an autotrophy marker:
- Wash the cells with with 1 ml of sterile water;
- Resuspend in 200 ?l and plate on selection plates;
If the transformation involves an antibiotic resistance marker:
- Resuspend the cells in 1 ml of YPD medium and leave for 1-2 hours at 28°C with agitation;
- Pellet the cells;
- Resuspend in 200 ?l and plate on selection plates.